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Single-cell Sequencing Nears Tipping Point in 2013


In 2013, single-cell sequencing dominated much of the research space in next-generation sequencing, driven by advances in technology and dropping costs of sequencing.

According to a report by market research firm DeciBio, the single-cell genomics market as a whole is expected to more than triple by 2015, with next-gen sequencing representing around $100 million of that in 2015 and growing to $300 million by 2018.

Evidence of this growth could be seen in the numerous peer-reviewed studies published in the year that advanced methods for sequencing single cells, including improving on whole-genome amplification techniques, automating the front and back ends of the workflow, and improving the sample prep process to increase yield. These improvements in the research setting are translating into commercial activity and successes particularly in the fields of oncology and prenatal genetic diagnosis.

In late 2012, single-cell sequencing research got a boost from the National Institutes of Health when it awarded groups from the University of California, San Diego, the University of Pennsylvania, and the University of Southern California five-year grants between $9 million and $10 million each under its newly established Single Cell Analysis Program to develop single-cell transcriptome sequencing methods.

The trend picked up throughout 2013 and included industry investment alongside academic research. Early in the year, Fluidigm launched its C1 system and protocols to automate the processing of cells for single-cell transcriptome sequencing, making the technology more accessible. The single-cell sequencing workflow involves complicated steps in cell isolation, sample preparation, and genomic analysis, and it is unlikely that any single researcher will be an expert in all three areas. When companies develop tools to address portions of these steps, such as cell isolation and sample prep, it opens up the technique to a broader range of genomics researchers.

Then late in the year, the company released a sample preparation workflow to early-access customers for the C1 system that processes single cells and amplifies their DNA for targeted, exome, and whole-genome sequencing.

Much of Fluidigm's technology is based on microfluidic techniques developed in the laboratory of Stanford University's Stephen Quake, a pioneer in the field of single-cell sequencing.

Fluidigm's microfluidic technology aims to address the challenges of isolating and sorting single cells, targeting researchers looking to sequence the genomes of multiple cells at once.

Chinese startup Yikon Genomics, however, is addressing a different aspect of single-cell sequencing — whole-genome amplification. Whole-genome amplification tends to be error prone. Different regions of the genome amplify at different efficiencies, a problem that is magnified when working with the minute amount of DNA from a single cell.

Yikon Genomics last year began providing single-cell whole-genome amplification, single-cell transcriptome amplification, and a variety of next-gen sequencing services. The firm licensed a whole-genome amplification technique known as MALBAC, for multiple annealing and looping-based amplification cycles developed by Harvard University's Sunney Xie, a cofounder of Yikon. The firm sells MALBAC-based whole-genome amplification kits and plans to launch a single-cell transcriptome amplification kit.

MALBAC aims to address the challenges of whole-genome amplification by using quasilinear rather than exponential amplification to enable more uniform amplification.

Within academia, much of the research in the last year has focused on addressing the problems of amplification bias.

Researchers from the Ludwig Institute for Cancer Research and the Karolinska Institute in Sweden have been working on a protocol dubbed Smart-seq, a single-cell RNA sequencing protocol that builds on template switching technology developed and patented by Clontech as SMART for Switching Mechanism at 5' End of RNA Template.

According to those researchers, template switching provides better coverage across the entire length of a transcript than other approaches. Originally published last year in Nature Biotechnology, the researchers have since improved upon the method in order to sequence more of the RNA in individual cells and generate longer RNA transcripts. The group published Smart-seq2 last fall in Nature Methods.

Separately, researchers from the University of California, San Diego have investigated the use of microwells to reduce amplification bias.

The technique, dubbed MIDAS, for microwell displacement amplification system, uses tiny nanoliter-sized wells to perform multiple displacement amplification — the whole-genome amplification technique most commonly used for single-cell sequencing.

The UCSD team demonstrated in a study in Nature Biotechnology that the method could sequence an entire Escherichia coli genome, recovering 50 percent more of the genome with three- to 13-fold less sequencing data compared to the most complete, published single-cell E. coli genome. Additionally, they showed that the method could identify copy number variations in single human neurons.

While it is unclear exactly why performing the MDA in tiny wells reduces bias, one hypothesis is that bias is reduced because there are fewer reagents for the same amount of starting material, preventing regions that naturally tend to amplify more quickly than others from growing as fast, leaving the slower-to-amplify regions time to catch up, according to the UCSD researchers.

Clinical applications

The advances made in single-cell sequencing began to enter the clinical realm in 2013, most notably in the fields of oncology and prenatal genetic diagnosis.

At last year's Advances in Genome Biology and Technology meeting in Marco Island, Fla., two research groups presented on their work using single-cell sequencing in in vitro fertilization to improve success rates by screening embryos before implantation for chromosomal abnormalities, a common cause of IVF failure.

Dagan Wells at the University of Oxford has devised a method that uses Life Technologies' Ion Torrent PGM to whole-genome shotgun sequence a single cell from the blastomere stage of an embryo to identify chromosomal aneuploidies.

He has so far tested it on 50 known samples, including 10 from known aneuploidies and 40 embryos that had been previously diagnosed with array CGH. All 50 samples yielded a result, with the 48 aneuploidies correctly called.

Wells has also been collaborating with Reprogenetics, a New Jersey-based firm that specializes in preimplantation genetic diagnosis.

Researchers from Peking University Third Hospital have been testing the MALBAC method in IVF applications, except they are focused on sequencing the polar bodies, which are byproducts of the IVF cycle.

In a recent study in Cell, the group demonstrated its protocol on oocytes from eight healthy volunteers and fertilized via intracytoplasmic sperm injection, sequencing the first and second polar bodies and cells from the blastocyst, for a total of 183 cells.

The group is now in the process of recruiting for a clinical trial to test the protocol in around 30 women who are either carriers of known Mendelian diseases, are more than 30 years old, or have experienced recurrent miscarriages.

Meantime, BGI is already using single-cell sequencing to screen for aneuploidies prior to IVF. The Chinese genomics firm is using a method published last year in PLoS One for detecting copy number variants from single-cell, low-pass, whole-genome sequencing.

BGI's Fei Gao presented on the firm's work at the Beyond the Genome conference in San Francisco in October.

In August, the first IVF baby that was sequenced before implantation was born healthy, Gao said, and since then more than 20 healthy babies have been born healthy following pre-IVF single-cell sequencing to screen for aneuploidies and large copy number variants.

Aside from reproductive health, the technique is also being used in oncology to sequence circulating tumor cells and to study tumor heterogeneity by sequencing single cells from different regions of a primary tumor.

For instance, James Hicks' laboratory at Cold Spring Harbor Laboratory is using a technique originally developed by Nicholas Navin called single-nucleus sequence and later refined to Cell-Seq, to sequence CTCs, monitor cancer cell trafficking, and to study cancer stem cells.

Hicks, who also reported at the Beyond the Genome meeting last fall, is using the technology to follow prostate cancer patients in real time to identify biomarkers indicative of drug resistance or sensitivity.

Single-cell sequencing of circulating tumor cells is especially useful for prostate cancer, he said, because the disease often metastasizes in the bone and there are frequently hundreds of metastases throughout the body, all of which cannot be biopsied.

Harvard's Xie is also studying CTCs in cancer patients using his MALBAC amplification method. In December, his group demonstrated in a study in the Proceedings of the National Academy of Sciences that the protocol could evaluate copy number variants in CTCs from lung cancer patients.

While many advances in single-cell sequencing techniques were made this year, and the technology is now advancing to the clinic, researches agree that there is still much room for improvement. As Xie said at the Beyond the Genome conference last fall, "there are high error rates in all the methods. … None of them are good enough."

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